GSM6931577: 91_20180727_7_sample2_IL13; Mus musculus; RNA-Seq
We obtained pooled mesentery from 20 wildtype C57BL/6 mice. Lineage-negative cells were selected by negative selection using MicroBeads and AutoMACS (Miltenyi Biotec GmbH). Then, c-Kit+Sca-1+ cells were sorted as ILC2s using a FACSAria flow cytometer (BD Bioscience). cDNA libraries were prepared using SMART-Seq v4 3'-DE Kits (Clontech) according to the manufacturer's instructions, with some modifications. A total of 3,892 copies of ERCC spike-in RNA were added to each sample as a mixture of quantitative internal controls. The cDNA was purified using Agencourt AMPure XP magnetic beads (Beckman Coulter). Library quality check was performed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and Agilent High Sensitivity DNA Kits (5067-4626). Degraded or low-yield samples were removed. Qubit High Sensitivity assays (Thermo Fisher Scientific) were performed to quantify the cDNA in each library, and 5 or 6 libraries with different indexed primers were evenly pooled. Pooled cDNA (400 pg) was tagmented using Nextera XT DNA Library Prep Kits (Illumina, San Diego, CA, USA), as described in the protocol. Library size and cDNA amount were quantified using an Agilent High Sensitivity DNA Kit and Qubit High Sensitivity assays, respectively.